Description

Biofilm is a community of microorganisms of same or different species encased in a self -produced extracellular polymeric substance on both living and non-living surfaces. Catheters are generally critical indwelling medical devices commonly used in clinical applications to enhanced flow of flu ids out of the patient’s body as well as influx of medications into human biological systems. Like many other indwelling medical devices, catheters are prone to enhanced risk of nosocomial infections which becomes a critical challenge as a result of microbial attachment to the surfaces of the catheters.
Attachment and subsequent biofilm development on catheter surfaces cause a number of persistent infections. The biofilm development increases resistance to antibiotics. However, this sometimes led to high pathogenesis, patient morbidity and mortality. This condition usually calls for a premature catheter removal which will increase the cost of treatment and improvidence of resources. This review focuses on how catheters get infected, microbial diversity among catheter biofilms, factors mediating biofilm formation on catheters and current strategies us ed in controlling biofilm formation on indwelling catheters.

214 JibrinNdejiko M ohammed et al.: Bacterial Biofilm: A M ajor Challenge of Catheterization the indwelling medical devices resulted fro m adhesion and subsequent capsule formation by the adherent organisms [7].Catheters are plastic tubes used to enhanced flow of med ications and waste substances into and out of the human system. Intravascular catheters are mainly used to pass med ical substances or medications directly to the patient’s blood circulatory system; urinary catheters on the other hand are used to remove waste fluids from the patient’s urinary compart ments[8]. The major challenge of catheterizat ion in health care services has direct relationship with development of biofilms on the catheters[9]. Biofilms can formed on indwelling med ical devices including prosthetic heart valve, pacemakers, central venous catheter, urinary catheter, contact lenses, intra-uterine devices etc. thereby causing persistent and deadly infections[10]. In another development, Infection of patients been treated with indwelling medical devices dated back to the fourteenth Century[11]. Meanwhile, the relat ionship between such infections and the Kosch’s postulate in many instances are still yet to be fully established thus imp licating biofilm as the causes of the indwelling device-related infections[9]. 2. General Overview of Biofilm Formation Biofilm is a co mmun ity of microorganisms of same or different species encased in an ext racellular poly meric matrix and are normally found on both living and non-living surfaces. Generally, biofilm format ion is initiated with the adherence of individual cells to the preconditioned biotic and abiotic surface layers[12, 13]. The presence of bulk solution or flu id on these surfaces serve as the basis or foundation for the biofilm formation[14, 15]. This is followed by a reversible attachment which is normally med iated by gravitational migration of indiv idual cells, motility of the bacteria and the shear force of the surrounding mobile phase[16]. The irreversib le attachment occurs as a result of concurrent production of the EPS and the strong interactive fo rces that exist between the microbial appendages and the surfaces[17]. This process occurs between 20 minutes to 4hours depending on the physiology of the species involved and the nature of the p reconditioned surface layer[17, 18]. Meanwhile, as the microbial cell move closer to a surface (<1n m), the in itial attach ment is facilitated by the attractive or repulsive electrostatic and hydrophobic interactions, steric hindrance and van der Waals forces[10]. The hydrophobic interactions have been postulated to play important roles in the primary adhesion[19], while the irreversible attachments are mediated by the molecular interactions that exist between specific adhesions and the surfaces[10]. The entire adhesion has also been exp lained with the aid of d ifferent mo lecular cell contact theories such as Derjagun-Landau-Verwey-Overbeck (DLVO) theory and the extended DLVO theory and the thermodynamic approach[14, 15, 20] The maturation of bio film is attained when the irreversibly attached cells develop to form a more organised and co mplex structure whose shape depend on source of the nutrients, however mushroom structure is common among bacteria biofilms[21]. This is followed by microbial dispersion which is the final stage of b iofilm develop ment and occurs when there is alteration in the mature biofilm structure as a result of increased shear force, depolymerisation of the EPS, use of antimicrobials and nutrient depletion[20, 22]. This final dispersion of biofilm is a complex process that plays a crucial role in in itiating and transmitting infect ious diseases. The major steps of the biofilm format ion are illustrated in the Figure 1 belo w as adopted from Otto, 2004. Figure 1. Themajor steps in Biofilm formation[23]

Journal of M icrobiology Research 2013, 3(6): 213-223 3. Contamination of the Catheters Over 80,000 in fections associated with catheters are been reported per annum by the intensive treatment units (ITU) of the United State of A merican (USA) alone and approximately 250,000 to 500,000 clin ical cases per annum in the entire hospitals in USA[24]. These catheter-linked infections orig inate fro m microorganisms that attached to the implanted device in the process of insertion, cross-infection via body contacts and/or during surgery with contaminated surgical tools. (See Figure 2) It has been observed that the principal waysby which microorganis ms reach and subsequently contaminate these catheters is usually through themovement of the skin’s microbial flora fro m the point of insertion to the catheter tract and along the tip of the catheter[25], Contamination of the catheter hub as a result of its direct contact with hands or contaminated liquids and other medical instruments, spreading of the contamination fro m another point of infection via blood flo w and rarely contamination as a result of an infusate. There have been well recognized evidences that associate catheters with infect ion and subsequent involvement of biofilms in such infections in various literatures. This is due to the fact that catheter itself is a 215 potential substratum for microbial adherence and subsequent biofilm development. Apart fro m being infected by the haematogenous route catheters can also serves as the channel for the passage of skin microbial flora into the patient’s body[8]. The Intravenous catheters and urinary catheters mostly used in many hospitals have also been found to be the frequent cause of nosocomial acquired bloodstream infections by the coagulase-negative staphylococci which account for about one third of episodes of catheter-related bloodstream infection[26, 27]. Infection of the Central venous catheter (CVC) has been reported to cause an in-hospital mortality rate of up to 35%, long time hospitalizat ions, and many complicat ions that are associated with the treat ment of such infections[8]. Catheter-associated urinary tract infections (CA UTIs) are also common type of hospital acquire infect ions that are of significant interest due to their complexity and frequent reoccurrence. These infect ions have been linked with Escherichia coli and Proteus mirabilis whose virulence factors have been reported to include adhesion, motility, biofilm format ion, resistance to immunity and acquisition of nutrient[28]. Figure 2. Schematic flow of catheter contamination and other events that lead to infection

216 JibrinNdejiko M ohammed et al.: Bacterial Biofilm: A M ajor Challenge of Catheterization 4. Role of Biofilms in Producing Catheter Related Infections The role of biofilms in in itiating catheter related infect ions cannot be overemphasized. Their roles include detachment of the cells fro m the b iofilm to in itiate b lood stream infections and infections of urinary tract, production of endotoxins by the biofilm encased gram negative bacteria [29], prevention of the damaging effect of the host immune system, transfer of p lasmid through the process of conjugation which leads to generation of antibiotic resistance strains[10], encrustation or obstruction of catheters with crystallised substances produced by the bacteria. Develop ment of gravels and pyelonephritis has also been associated with bacterial colonization of the catheters and subsequent biofilm formation[30]. Rodríguez et al.[31] investigated Bio film formation in Acinetobacterbaumannii with respect to its associated features and clinical implications using microtiter assay and reported biofilm formation by Fifty-six (63%) of isolated 92 strains. Thomson et al,[8] also reported that urinary o r b loodstream infections associated with catheter and cerebral shunt-related men ingitis are caused by bacterial strains that have potential to form biofilm easily. In another develop ment Do lan (2001) stated in his rev iew that biofilm formation on CVCs is universal, He however emphasized that the extension and location of such biofilm is dependent on the length of catheterization. Short-term catheterization (<10 days) tends to favour greater b iofilm development on the external surface while long-term catheterizations (30 days or more) tend to favour greater biofilm growth on the inner lu men of the catheter[32]. It is also important to note that the accidental breakdown of catheterization can result in early bacteria colonization of the catheters. Evaluation and analytical co mparison of intra lu minal and extra-lu minal source of urinary catheter-related infections revealed that unexpected breakdown of catheters and subsequent introduction of contaminants causes bacteriuria within first two days. However, bacteriuria delayed to after 3-7 days when closed and sterile catheterization was maintained[33]. The type of the flu id passing through the catheters can also influence biofilm fo rmation in the sense that Gram-positive organisms such as Staphylococcus epidermidis and Staphylococcus aureusgrow poorly in intravenous fluids, whereas Gram-negative bacteria such as P. aeruginosa, Pantoeaspp, Klebsiellaspp., Enterobacterspp. and Serratiasppcansustained their growth in intravenous fluids [32]. Upon insertion, both the inner and outer surfaces of the urinary catheters serve as targets for microbial attachment and subsequent biofilm development. Endogenous microorganis ms that originate fro m the gastrointestinal tract have been found to colonized the region of the body between the anus and the scrotrum/ vulva and ascend the catheter via extra-lu minal route during catheter insertion. The exogenous microorganis ms which can originate fro m cross contamination fro m the hands of medical personnel on the other hand, move upward the catheter via intra-lu minal route (access of the organisms to the inner lumen of the catheter) [34]. Investigation of a number catheters shows that the thickness of catheter biofilms ranges fro m small patchy layers of attached cells to extensive biofilms that often covers the entire length of the inner lu men of the catheter and comprising of a very high bacterial population[7] 5. Microbial Diversity in Biofilms of the Catheter Over the last decade, catheters, most especially the urinary catheters has become the second commonly used indwelling material inserted to the body of patients. This has subsequently resulted in increased infections of urinary catheters (IUC) with over 40% of the nosocomial infections occurring in catheterized patients during the first 10-14 days of catheterizat ion[30]. Microrganisms most often isolated fro m catheter biofilms include Candida albicans, P. aeruginosa, K. pneumoniae, Enterococcus faecalis, S. epidermidis[32], Acinetobacterbaumannii[8] and Escherichia coli, while the strongest biofilm producers are Proteus mirabilis, E. faecalis, Candida tropicalis, andStaphylococcus aureus which are also responsible for the mixed species biofilms[30]. Bonkatet al[35] studied microbial b iofilms in suprapubic catheterisation that are nowadays used as an alternative to urethral catheterization using conventional culture techniques and isolated a total of 428 microorganisms of wh ich Enterobacteria, Enterococcus and P. aeruginosa were the most frequently isolated spp. They also compared the frequency of catheter associated with bacteriuria in suprapubic catheterisation with those of published findings of urethral catheters[36-38] and reported no significance difference between the two. They were however speculative on the reduction of catheter associated urinary tract infection with the suprapubic catheterization [35] . In another development, Choeet al[39] analyzes bacteria distribution of biofilms that grows on urinary catherters using 4 different 16S rRNA approaches namely capillary electrophoresis, terminal restriction frag ment length polymorphis m (T-RFLP), denaturing gradient gel electrophoresis (DGGE), and pyrosequencing. They isolated 329 spp of which Edwardsiella, Enterobacter, Escherichia, and Pseudomonas were the predo minant isolates for each of the four techniques. Other represented organisms they isolated include Moraxella, Proteus, Serratia, Yersinia, Burkholderia, Corynebacterium, Achro mobacter, Alcaligen es, Citrobacter, Stenotrophomonas, and Streptococcus[39]. Meanwhile, systematic antimicrobial treat ments during catheterization have been reported to reduce microbial diversity on the urinary catheters. Frank et al.[40] used mo lecular techniques involving PCR and cloning to investigate diversity of microorganis ms on the urinary catheters and reported that both outer and inner surfaces of 8 catheters removed fro m patients undergoing no systemic

Journal of M icrobiology Research 2013, 3(6): 213-223 antimicrobial treat ment were colonized by 20 different microbial species. However a wide variation existed between the internal and outer surfaces whereas only one catheter out of six was colonized by a single microbial species in patients undergoing systematic antimicrobial treat ment[40, 41]. 6. Factors Mediating Biofilm Formation on Catheters Develop ment of bio films on catheters is determined by several variables. The microorganis ms must be able to adhere to the exposed surface of the catheter and become irreversibly attached. Attachment of the microbial cells is dependent on the physiological and chemical properties of the surface, hydrophobicity of the microorganisms and the substratum; catheters that are both hydrophobic and hydrophilic in nature favours attachment of wide species of microorganis ms. Divalent cations such as calcium and magnesiu m increases the urinary pH and ionic strength and has been reported to facilitate bacterial attach ment[42]. Other factors include production of platelets, t issue proteins and sera (host preconditioning films), nu mber and type of the microbial cells, the rate at which the liquid flows through the surface of the catheter and the composition of the liquid itself[15, 43, 44]. As a result, the irreversible attachment and production of EPS pro mote the format ion of bio film as the biofilm growth rate continue to be affected by the flow rate, nutrient and antimicrobial co mposition of the liquid passing through the catheter and the ambient temperature[32]. In another development, the surface coating of the central venous catheters with heparin has been reported to exhib it the potential to reduce in vitro and in vivo microbial attachments. Appelgrenet al. (1996) found that in v itro adherence of coagulase-negative Staphylococci to heparin coated catheters was drastically reduced as compared to non heparinized catheters (p < .05). In the in vivo study of 32 central venous catheters, he observed bacteria and fungi colonizat ion in only four o f the thirteen heparin ized catheters while fourteen out of nineteen were colonized in the no heparinised catheters with the coagulase-negative Staphylococci been the most frequently isolated bacteria in both cases[45]. It has however been observed that low citrate concentration and sodium heparin that is widely used as catheter lock solution can serve as a strong stimulant for S. aureus biofilm format ion[46]. 217 urinary catheters[47]. In addition to occluding the lumen of the catheter and subsequent urine leakage or retention which inflicts pains and distress on the patients[48], these deposits also causes trauma to the urethra and bladder mucosa. Encrustation and unidentified blockage can also lead to disease conditions such as pyelonephritis, septicaemia and shock[47]. About half of catheterized patients most especially, the elderly people suffer fro m encrustation and obstruction (catheter blockage). This is co mmon o f long time catheterization and gives rise to more co mmun ity nursing caseload[49]. A wide number of studies have demonstrated that the steps involved in encrustation to include bacteria contamination of the urinary tract p redominantly by P. mirabilis and other urease producers, mult iplication and their attachment to the catheter, subsequent biofilm format ion and accumulat ion of the extracellular poly mers which elevate the urine pH and triggers the stabilization of the crystallized magnesiu m and calciu m phosphates on catheter inner surface[48, 50-52] Electron microscopy of catheter encrustation reveals the presence of large nu mbers of bacilli known to easily form biofilm conforming to the view that encrustations originate fro m mineralized b iofilms. Bacterio logical studies of the encrusted catheter biofilms also show the urease producing P. mirabilis to be the dominant isolate[7, 47]. A lthough many studies reported biofilm mineralisation to be involve in encrustation, other studies reported involvement of bacterial capsule polysaccharide wh ich enhance more stabilization of the crystals[51, 53]. Thus the general methods for preventing encrustation involves incorporating antimicrobials to the polymers and designing catheters whose surface property will not allow in itial bacterial attach ment[54]. A photograph of catheter tip with encrustation (adapted fro m Hoet al.) is shown in Figure 2. Figure 3. Residual encrustations at the catheter tip after it removal[55] 7. Encrustation and Obstruction of the Catheters by Biofilms Encrustation of catheters results from the deposition of mineral salts such as magnesium phosphate, ammoniu m phosphate and calciu m phosphate on both the inner and outer layer of the catheters. These mineral salts accumulate as a result of microbial activ ities that exert functional roles to increase the acidity of normal urine and turn it alkaline in 8. Mechanism of Antimicrobial Resistance in Catheter Biofilms Generally, antimicrobial agents known to be effective against microorganisms that grow in suspension frequently fail to exert their effects when used on the microorganisms that grow in biofilms. Th is has been attributed to a variety of mechanis ms wh ich includes the accumulation o f the

218 JibrinNdejiko M ohammed et al.: Bacterial Biofilm: A M ajor Challenge of Catheterization extracellular poly meric substance that tends to allows partial or zero penetration of the antimicrobial agents in to the microorganis ms (see Figure 4) for instance,aminoglycosides with positive charge can bind to negatively charged polymeric substances in the biofilm matrix and retard their penetration. EPS can also dilute the antimicrobial concentration before they get to the individual cells in the biofilm, thus reducing the potency of the antibiotics against microorganis ms[4, 56]. M icroorganisms grow slowly when they are in biofilm and therefore become resistance to antimicrobial agents that requires vigorous microbial growth, the slow growth also promote poor expression of antimicrobial binding proteins among organisms growing in biofilms. Other mechanis ms of resistance in biofilms include activation of mult iple genetic materials that allow microorganis ms to alter their cell envelop, the molecular targets, and the susceptibility to specific antimicrob ials. This mechanis m of resistance is otherwise refers to intrinsic resistance[57, 58]. Figure 4. Mechanisms of biofilm antimicrobial resistance[64] Similarly Stewart and Costerton[58] reported three mechanis ms of antimicrobial resistance in biofilms: i. Slow penetration in which antibiotics may not be able to move beyond the surface layers of the biofilm ii. Develop ment of resistance phenotypes which allo ws the bacteria growing in bio film to differentiate in to protected phenotypes see Figure 4 also iii. Altered microenvironment in which the antibiotic actions are antagonised as result of zones of nutrient accumulat ion or waste accumu lation An increased expression of the efflu x pu mp is another mechanis m that decreased susceptibility of biofilms to antibiotics. Studies of b iofilm format ion by P. aeruginosa, uropathogenicE. coli and Candida albican revealed the specificity of the upregulation of genes that encodes antimicrobial transporters or the factors that regulates such transporters[59, 60]. However, investigation of biofilm global gene expression indicated that upregulation of antibiotic transporters is not universal and therefore only restricted to antibiotics that possesses efflu x transporting substrates[59]. The continuous application of chlorohexid ine for the management of long-term bladder catheters has resulted in the development of chlorohexidine-resistant bacteria[61] which were later found to be resistant to many other drugs[62, 63]. The isolated resistance strain, Proteus mirabilis has been identified to be responsible for pyelonephritis, bladder and kidney stones and the encrustation and obstruction of catheters in urinary tract catheterizat ion[61]. 9. Current Strategies in Controlling Biofilm Development on Catheters One of the best ways to prevent biofilm development on any surface is to prevent the initial attach ment of microbial cells by avoiding contamination of the exposed surface. The preventive measures in catheter related blood stream infection include prevention of extra-lu minal contamination through adoption of aseptic measures such as the use of sterile gloves, gown, cap, and mask during the insertion of the catheters. The use of skin antisepsis such as 2% aqueous chlorohexidine-containing antiseptics which has been reported to be very effective is also another pre-infection measure[65]. The nu mber of skin micro flora at the insertion site is an important factor that should be considered to prevent catheter related blood stream infect ion. It is recommended that central venous catheters be inserted at sub-clavian sites as the catheters inserted into jugular have been reported to possesses higher risk of microbial colonizat ion in comparison to those inserted into a subclavian[25, 66]. Furthermore, to prevent endoluminal contamination, the hub should be handled with high degree of asepsis. This can be achieved by protecting the hub using an iodine-impregnated foam or povidone, external p rotection of the hub, ensuring spacing in changing the infusion set and reduced number of lu mens in the catheter. Povidone iodine is also the commonly applied antiseptic in USA for cleansing arterial catheter and insertion site of the central venous catheters (CVC)[67]. Other preventive measures include avoiding unnecessary manipulation of catheters, use of antiseptic connectors and involvement of well-t rained proffessionals[66]. So me of the common ly used strategies of preventing contamination of the urinary catheters such as systematic or direct introduction of antimicrobial agents in to the bladder and catheter irrigation has all been reported to be ineffective measures of preventing microbial colon izat ion of the urinary catheters[36]. Modification of materials used for catheter design is another economic and effective preventive measure that is recently used in the medical industry. In this approach, the catheter surfaces are modified to avoid microbial adhesion[68, 69]. The proposal is to construct a device without any fouling properties in o rder to min imize adsorption of protein and subsequent microbial adhesion and at the same time preserving the favourable characteristics of the device in terms o f its strength and inertness[70]. An in vitro study has shown that use of heparin, sodium cit rate and sodium EDTA, can hindered biofilm development by S. aureus indicating that they can be applied to reduce the biofilm-associated infections in indwelling catheters[46]. Furthermore, the used of antimicrobial agents to control

Journal of M icrobiology Research 2013, 3(6): 213-223 biofilm format ion on medical indwelling devices has been investigated by many researchers. Dolan[32] rev iewed that augmentation of dextrose-heparinized left atrial catheter with sodium metabisulfite prevented the microbial colonizat ion of the catheters and also reported that minocycline and rifampin imp regnated catheters experiences less microbial colonization as compared to chlorohexidine and silver sulfad iazine imp regnated catheters. Similarly, the impregnation of central venous catheter with minocyclinerifampin (M/R CVCs) has been found to be efficient in combating catheter-related blood stream infect ions and subsequent biofilm formation by both Gram-positive and Gram-negative organisms[71, 72]. It was however not effective on P. aeruginosa and Candida spp.In order to increase the antimicrobial performance of this approach, Raadet al.[71] developed a novel minocycline/ rifamp in catheter that included chlorhexid ine (CHX-M/R catheter) which they found to have prolonged activity and effective in complete inhibit ion of all the resistance strains including the Pseudomonas aeruginosa and Candida spp. In a related research, Kamal et al.[73] in their study of reduced intravascular catheter infection by antibiotic bonding found that cationic surfactant coated catheters bonded with cephalosporine has less microbial contamination and biofilm development than the untreated catheters. Application of ointment containing mu ltiple antibiotics prior to insertion of attachable subcutaneous cuff that contains silver ions, coating the inner lumen of the catheter with an antibiotic, using topical antibiotics and removing the contaminated catheters can also reduce contamination of catheters and biofilm development that acco mpany such contaminations [32]. Dolan[74] reviewed that novel techniques such as the use of chelating agents, biofilm d ispersants, quorum sensing inhibitors and bacteriophages can eradicate biofilms on intravascular catheters. The Chelating agents such as ethylene diaminetetraacetic acid tetrasodium EDTA or disodium EDTA and minocycline-EDTA are capable of destabilizing the biofilm structure[75]. To support this, Percival et al.[76] and Kite et al.[77] reported that 40 mg/ mL of tetrasodium EDTA was able to eliminate biofilms in an in vitro model and on hemodialysis catheters respectively. Other forms of preventive techniques include: i. mixture o f t igecycline and disodium EDTA with gentamicin and disodium EDTA which has been reported to drastically reduced biofilms of Staphylococcus species and P. aeruginosa growing on Hickman catheter[74]. ii. Dispersal of microbial cells fro m b iofilms by shedding of daughter cells during active growth can be achieved by changing the nutrient levels or quorum sensing or by the use of flow shear force. iii. Oxid izing biocides, such as chlorine, surfactants, or enzy mes can also cause disruption and subsequent dispersal in biofilms[74]. iv. Unsaturated fatty acids such as cis-2-decanoic acid produced by P. aeruginosa can disperse several clin ically relevant biofilms in vitro. This type of dispersion is taught to 219 be as a result of degradation of the EPS by neighbouring cells in response to the cis-2-decanoic acid (signalling mo lecule). However this approach requires additional treatment with antibacterial agents to prevent reattachment of the dispersed cells[78]. v. So me strains of bacteriophage produce polysaccharide depolymerases that are capable of degrading the biofilm EPS. Curtin and Dolan[79] and Fu et al[80] both studied the use of bacteriophage to prevent biofilm p revention and reported the prevention ofS. epidermidisand P. aeruginosabiofilm growth on phage treated catheters. 10. Techniques of Studying Catheter Biofilms Generally, a wide nu mber of techniques and models have been used to study microbial biofilms on different surfaces. Some of the co mmon techniques include d irect microscopic techniques such as confocal laser scanning microscopy, atomic force microscopy and scanning electron microscopy, micro man ipulation[1, 14, 81-83]. These models involves theuse of flow cells, 96 well micro titer plate also refered to as the Calgary Biofilm Dev ice, colony biofilms, biofilm ring test, micro fermentors and modified robbins devices[84, 85]. However, so me of these models yielded positive results in in vitroinvestigation, they do not really rep resent the ideal conditions in the studies of bio film related infection such as catheter biofilms.In order to establish a comprehensive technique for studying and controlling biofilms on medical instruments such as catheters, there is need for simu lation and development of direct and no destructive techniques and models that represent the actual clinical conditions[86]. In order to quantify biofilms on central venous catheters, an internationally referenced method otherwise known as Maki's semi-quantitative method or roll plating technique can be employed. In this method, the tip of the catheter is removed and moved randomly over a general purpose agar med iu m to count the number of the microorganis ms after a specified period of incubation[32]. This method suffers the disadvantage of not able to discover more than 1× 103 colonies and it inability to detect biofilms gro wing on the inner lu men of the catheter[87]. An imp roved semi quantitative roll plat ing that uses sonication and vortex to quantify biofilms can detect up to 1 × 104 colonies per tip however there is need for further determination of its recovery efficiency[86]. The use of acrid ine orange to directly stains the catheter biofilms is a rap id method that do not modify the clinical conditions of the catheters and simp ly record positive and negative results instead of quantifying the cells[32, 88]. Another culture independent technique used for studying catheter biofilms is the molecu lar technique which involves 16s rRNA, poly merase chain reaction PCR, denaturing gradient gel electrophoresis DGGE and Fluorescence in situ hybridizat ion FISH[39, 89, 90]. These techniques is based on application of PCR to amp lify the seg ment of 16s rRNA isolated from the biofilm sample, this will produced a

220 JibrinNdejiko M ohammed et al.: Bacterial Biofilm: A M ajor Challenge of Catheterization combined PCR products from the various microorganisms that constitute the biofilm. The PCR products are subsequently subjected to screening and separation by DGGE to produce an order of bands which correspond to various microbial species in the biofilm sample[89]. This technique is faster and overcomes the problem of misrepresentation in the rolled plate culture technique and has been used to study microbial d iversity of biofilm samples [39, 89]. In another development, Hassan et al[91] co mpared Tissue Culture Plate (TCP) method, Tube method (TM) and Congo Red Agar method (CRA ) for their ability to detect biofilm formation in about 110 clin ical isolates and reported the superiority of the TCP over TM and CRA, for the details about these methods the reader is refer to the work of Hassan et al[91]. In a related study, clement et al.[92] used crystal violet staining, biofilm ring test, and resazurin assay to study biofilm fo rmation in 34 clinical E. coli strains. Co mparing the analysis of the 3 methods, they state that “there was significant correlation between CV and RZ assay (Spearman r = 0.68; P < 0.0001) and between CV and BRT (Spearman r = 0.54; P = 0.0007). RZ and BRT were not significantly correlated (Spearman r = 0.18; P = 0.28)”. 11. Conclusions Microbial biofilms pose a major challenge to the entire catheterization process and account for most nosocomial infections in catheterized patients or patients under treatment with other indwelling medical devices. Apart from resistance to host immune system, the developments of biofilms by the microorganis ms drastically reduce their sensitivity to the antimicrobial agents and make them almost impossible to eradicate using the conventional methods. In addition to obstruction and encrustation of the catheter by these biofilms which inflict serious pains and distress on the patients, detachment of microbial cells fro m the catheters results in serious infections. Although medical indwelling devices has different design features, important factors such as duration of catheterizat ion, co mposition of the surrounding fluids or nature of fluid flowing through the catheter, the flow rate, the type of the contaminating microorganism and preconditioned film determines the type and extent of biofilm develop ment on indwelling catheters. Combating the challenge posed by biofilm develop ment on indwelling catheters is based on conducting researches that evaluate the existing control strategies and their effectiveness while developing both in vivo and in vitro catheter related models of biofilms that considers the specific conditions found in catheters in order to come up with reliable and novel techniques. Development of novel aseptic measures that prevents the initial colon izat ion and microbial attachment to the devices will also help in co mbating the challenges of biofilms in medical care. There is also need to improve the in situ imaging of biofilms, probes for real time analysis and characterizat ion of biofilm specific gene regulators. REFERENCES [1] Pamp, S.J., C. Sternberg, and T. Tolker‐Nielsen, Insight into the microbial multicellular lifestyle via flow‐cell technology and confocal microscopy. Cytometry Part A, 2008. 75(2): p. 90-103. [2] Nielsen, M .W., C. Sternberg, S. M olin, and B. Regenberg, Pseudomonas aeruginosa and Saccharomyces cerevisiae biofilm in flow cells. Journal of visualized experiments: JoVE, 2011(47). [3] Flemming, H.-C., Biofilms and environmental protection. Water Science & Technology, 1993. 27(7-8): p. 1-10. [4] Bose, S. and A.K. Ghosh, A Challenge To M edical Science. Journal of Clinical and Diagnostic Research, 2011. 5(1): p. 127-130. 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